Immune Responses to Hepatitis C Virus Structural and Nonstructural Proteins Induced by Plasmid DNA Immunizations

Gordon, Ethel J.; Bhat, Ramesh A.; Liu, Qing‐Yan; Wang, Yun-Fen; Tackney, Charles; Prince, Alfred M.

doi:10.1086/315162

Cited by 42 publications

(25 citation statements)

References 39 publications

(40 reference statements)

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“…Splenocytes from vaccinated mice were pulsed with 10 g of Core, E1, or E2 peptide per ml, corresponding to HCV genotype 1b, which has been previously shown to activate CTLs in immunized BALB/c mice (27,53). As shown in Fig.…”

Section: Vol 76 2002mentioning

confidence: 99%

Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector

Ezelle

Marković

Barber

2002

J Virol

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Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.Hepatitis C virus (HCV), a hepatotropic, positive-stranded RNA virus of the Flaviviridae family, is estimated to infect at least 400 million people worldwide and is a major etiologic agent of liver failure and hepatocellular carcinoma (11,66,70). HCV comprises a 9.6-kb genome with a conserved 5Ј untranslated region that functions as an internal ribosome entry site (33, 69). The untranslated region precedes a long open reading frame that encodes a 3,010-amino-acid (aa) polypeptide that is subsequently cleaved into 10 protein products (33). The amino-terminal region of the viral polypeptide is posttranslationally processed by host cell proteases to generate three structural protein products, Core (nucleocapsid) and envelope glycoproteins E1 and E2 (31, 43). Nonstructural proteins that facilitate virus replication (NS2, -3, -4a, -4b, -5a, and -5b) reside in the carboxy region of the polypeptide and are cleaved by virus-encoded proteases comprising 68).Standard therapeutic intervention for HCV infection consists of the administration of interferon (IFN) in combination with ribavirin. However, less than 50% of infected patients respond to this regimen and few alternative therapies exist (13, 14, 26, 52). There is presently no tissue culture system to efficiently cultivate HCV, which not only hampers research efforts aimed at elucidating the molecular mechanisms of virus replication but also impedes attempts at producing candidate vaccines and immunotherapies that target HCV-related disease. Consequently, a number of recombinant subunit-based HCV vaccine strategies, involving genetic immunization and purified proteins, have been attempted, as well as virus vectors expressing the HCV envelope glycoproteins (3,8,25,30,56,63,65). Determining an effective vaccination strategy has proven di...

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Section: Vol 76 2002mentioning

confidence: 99%

Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector

Ezelle

Marković

Barber

2002

J Virol

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“…It has been shown that DNA immunizations can induce both specific antibodies and cell-mediated responses against the structural and nonstructural (NS) HCV proteins in mice. [6][7][8][9][10][11][12][13][14][15][16] The NS protein 3 of HCV has been considered as a possible vaccine target. The reasons for this are that the NS3 protein shows a limited genetic variability, it performs multiple enzymatic functions, and it is a relatively large protein.…”

Section: Introductionmentioning

confidence: 99%

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene

et al. 2004

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We have recently shown that the NS3-based genetic immunogens should contain also hepatitis C virus (HCV) nonstructural (NS) 4A to utilize fully the immunogenicity of NS3. The next step was to try to enhance immunogenicity by modifying translation or mRNA synthesis. To enhance translation efficiency, a synthetic NS3/4A-based DNA (coNS3/4A-DNA) vaccine was generated in which the codon usage was optimized (co) for human cells. In a second approach, expression of the wild-type (wt) NS3/4A gene was enhanced by mRNA amplification using the Semliki forest virus (SFV) replicon (wtNS3/4A-SFV). Transient tranfections of human HepG2 cells showed that the coNS3/4A gene gave 11-fold higher levels of NS3 as compared to the wtNS3/4A gene when using the CMV promoter. We have previously shown that the presence of NS4A enhances the expression by SFV. Both codon optimization and mRNA amplification resulted in an improved immunogenicity as evidenced by higher levels of NS3-specific antibodies. This improved immunogenicity also resulted in a more rapid priming of cytotoxic T lymphocytes (CTLs). Since HCV is a noncytolytic virus, the functionality of the primed CTL responses was evaluated by an in vivo challenge with NS3/4A-expressing syngeneic tumor cells. The priming of a tumor protective immunity required an endogenous production of the immunogen and CD8 þ CTLs, but was independent of B and CD4 þ T cells. This model confirmed the more rapid in vivo activation of an NS3/4A-specific tumor-inhibiting immunity by codon optimization and mRNA amplification. Finally, therapeutic vaccination with the coNS3/4A gene using gene gun 6-12 days after injection of tumors significantly reduced the tumor growth in vivo. Codon optimization and mRNA amplification effectively enhances the overall immunogenicity of NS3/4A. Thus, either, or both, of these approaches should be utilized in an NS3/4A-based HCV genetic vaccine.

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“…However, it is likely that the use of long inserts often leads to both a decrease of gene expression and instability of plasmids during amplification. Although it was reported that a DNA construct encoding a whole HCV polyprotein can induce HCV-specific T-cell responses, those T-cell responses were not directly compared to T-cell responses induced by a mixture of multiple plasmids with shorter-length inserts (16). One of the most critical concerns to be addressed is the relationship between insert length in a plasmid and the strength of induced T-cell responses.…”

mentioning

confidence: 99%

Optimal Induction of T-Cell Responses against Hepatitis C Virus E2 by Antigen Engineering in DNA Immunization

Youn

Park

Cho

et al. 2003

J Virol

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Immune Responses to Hepatitis C Virus Structural and Nonstructural Proteins Induced by Plasmid DNA Immunizations

Cited by 42 publications

References 39 publications

Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector

Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector

Codon optimization and mRNA amplification effectively enhances the immunogenicity of the hepatitis C virus nonstructural 3/4A gene

Optimal Induction of T-Cell Responses against Hepatitis C Virus E2 by Antigen Engineering in DNA Immunization

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